Determining the Effect of Deficient Neuropeptide Signalling on Circadian Rhythms and Behavioural Physiology in Mice.

  • Rayna Samuels

    Student thesis: Master of Philosophy

    Abstract

    All organisms show daily rhythms in endocrine output, physiological responses and behaviour. Many of these are endogenous rhythms which are under the control of circadian clocks. In mammals, the master circadian clock responsible for orchestrating these rhythms is located in the brain's suprachiasmatic nucleus (SCN). The SCN itself is composed of thousands of cellular oscillators and many SCN neurons produce the neuropeptide vasoactive intestinal polypeptide as well as its receptor, VPAC2R. Previous investigations using animals carrying the null mutation for either the VIP peptide (Vip-/- mice) or the receptor VPAC2R (Vipr2-/- mice) have established that VIP-VPAC2R signalling is important for synchronising these cellular oscillators of the SCN. Around 40-60% of Vip-/- and Vipr2-/- animals lack behavioural rhythms, but some mice do retain a degree of rhythmicity. In this study, Vip-/- and Vipr2-/- mice were bred together to yield mice completely deficient in both the functional VIP peptide and the VPAC2 receptor (Vip-/- x Vipr2-/- mice). Immunohistochemical examination of the SCN confirmed that immunoreactivity for VIP or VPAC2R was absent in these mice and that in comparison to the wild-type SCN, the distribution of other signalling neuropeptides of the SCN was not profoundly altered by the loss of VIP-VPAC2R signalling. Subsequent evaluation of running wheel behaviour under 12:12 (LD), constant dark (DD), or constant light (LL) and also during reduced dark phase cycles (18h light: 6h dark) was performed to determine the behavioural consequences of the loss of this neuropeptide signalling system. Under LD conditions, all WT and ~80% of Vip-/- x Vipr2-/- mice were behaviourally rhythmic. Under DD conditions, a small proportion (~30%) of Vip-/- x Vipr2-/- animals displayed rhythmicity in running wheel behaviour, while all WT animals were rhythmic. The initial onset of activity in these rhythmic Vip-/- x Vipr2-/- animals typically occurred ~8h advanced relative to that seen under the previous LD cycle, while that of WT mice was in phase to that of the earlier lights-off. This indicates impaired synchronisation to lights-off in Vip-/- x Vipr2-/- mice. To test how the SCN of Vip-/- x Vipr2-/- animals responds to transient light exposure, mice in DD were pulsed with 60 min of light during projected day or projected night and the expression of the immediate early gene product, c-Fos determined immunohistochemically. While the SCN of WT mice only showed elevated c-Fos following the light-pulse during projected night, the SCN of Vip-/- x Vipr2-/- animals showed elevated c-Fos to light pulses given at either day or night. This indicates that appropriate gating of photic input is lost in the SCN of Vip-/- x Vipr2-/- animals. To test whether periodic exposure to a non-photic Zeitgeber could promote rhythmicity in Vip-/- x Vipr2-/- mice, WT and Vip-/- x Vipr2-/- animals in constant dark were placed under a scheduled voluntary exercise (SVE) regimen for 21 days and their subsequent circadian competence determined. Although most Vip-/- x Vipr2-/-animals showed apparently synchronisation to the SVE stimulus, only a relatively small proportion of these mice sustained behavioural rhythms when the regimen was terminated. Therefore, unlike in Vipr2-/- mice, SVE cannot promote behavioural rhythmicity in most Vip-/- x Vipr2-/- animals. Finally, the neuropeptide signalling can influence metabolic activity and so metabolic rhythms and ingestive behaviour rhythms were assessed in Vip-/- x Vipr2-/- mice and compared to WT controls. A small proportion of Vip-/- x Vipr2-/- animals had detectable metabolic rhythms and these were significantly lower in amplitude rhythms than those seen in WT controls. These studies indicate that the complete absence of VIP-VPAC2R signalling leads to altered circadian responses to light and SVE and severely compromises the ability of the SCN to control daily rhythms in metabolism and behaviour.
    Date of Award1 Aug 2013
    Original languageEnglish
    Awarding Institution
    • The University of Manchester
    SupervisorHugh Piggins (Supervisor)

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